EPO and EPO Receptor (EPOR)
Erythropoietin (EPO) is the primary stimulator of erythroid progenitor cells. The role of recombinant human (rh)EPO in erythropoiesis is well characterized and has been known for more than 50 years (Jacobson et al., Trans. Assoc. Am. Physicians. (1957) 70:305; Muench and Namikawa, Blood Cells Mol. Dis. (2001) 27:377). The three-dimensional structure of EPO and the extracellular domain of the homodimeric EPOR has been determined (Syed et al., Nature (1998) 395:511). EPO binds to the conserved receptor homology (CRH) domain that shares structural features with a family of related cytokine receptors and includes two immunoglobulin-like beta-sheet domains. Upon binding to a predimerized receptor, EPO activates the JAK2-STAT5 signaling pathway resulting in activation of nuclear transcription (Witthuhn et al., Cell (1993) 74:227; Yoshimura and Misawa, Curr Opin Hematol. (1998) 5:171), and Jak2 phosphorylation is also critical for EPO's neuroprotective activities (Digicaylioglu and Lipton, Nature (2001) 412:641).
The GM-CSF/IL-3/IL-5 receptor common beta-chain (CD131) has been shown to functionally and physically associate with the extracellular portion of EPOR (Brines et al., PNAS (2004) 101:14907; Jubinsky et al., Blood (1997) 90:1867, and the tissue- and neuroprotective effects of EPO have been linked to the expression of CD131 (Brines et al., PNAS (2004) 101:14907; Leist et al., Science (2004) 305:239). No protective effects of EPO were observed in mice lacking CD131 (Brines et al., PNAS (2004) 101:14907). However, in vitro studies in rat cell line PC12 indicated that coexpression of CD131 is not always a prerequisite for the cytoprotective activities of rhEPO (Um et al., Cell Signal. (2007) 19:634). CD131 was also shown to associate with EPOR on endothelial cells, and CD131 was crucial for the anti-apoptotic effects of rhEPO in these cells (Su et al., J. Cell Physiol. (2011) 226; 3330; Bennis et al., J Thromb Haemost (2012) 10:1914). These data suggest that CD131 association with the “classical” EPOR is critical for both neurons and endothelial cells.
In selected indications, such as in organ protection and CNS diseases, EPO Receptor Agonists and EPO mimetic compounds that demonstrate low erythropoietic activity, while exhibiting EPO-like cytoprotective effects, will likely provide optimal efficacy while avoiding hematopoietic adverse events. Variants of rhEPO and EPO mimetic peptides with tissue- and neuroprotective activity in vivo and little or no hematopoietic activity have been described, further indicating that these functionalities are independently regulated. For example, carbamylated and desialylated derivatives of EPO and EPO mimetic peptides with apparent tissue protective activities induced only minor effects on reticulocyte counts in mice and rats (Brines et al., PNAS (2004) 101:14907; Erbayraktar et al. (2003) 100:6741; Leist et al. Science. (2004) 305:239; Swartjes et al., Anesthesiology (2011) 115:1084; Patel et al., Mol Med. (2012) 18:719; Robertson et al., J Neurotrauma. (2012) 29:1156). While all these new EPO variants and mimetic peptides have been designed to provide benefits over rhEPO, they will likely have the same limitations as rhEPO when it comes to pharmacokinetic properties, crossing the BBB and risk of immunogenicity. Non-peptidic EPO mimetic compounds have been reported, which bind to EPOR in the cytokine binding domain (Qureshi et al., PNAS (1999) 96:12156, Goldberg et al. J. Am. Chem. Soc., (2002) 124:544). Another screen demonstrated the efficacy of small heterocyclic agonists, which appear to act as EPO mimetics and acted in synergy with EPO in cellular proliferation assays (Olsson and Naranda U.S. Pat. No. 7,037,902).
EPO and EPO Mimetics in Tissue and Neuroprotection
A significant role of rhEPO and other EPOR Agonists in tissue- and neuroprotection and neurogenesis has emerged, suggesting therapeutic applications for organ protection, wound healing, transplantation and diverse neurological disorders, such as Friedreich's ataxia, stroke, multiple sclerosis, spinal cord injury, traumatic brain injury, schizophrenia, depression, Alzheimer's disease and Parkinson's disease (Brines et al., PNAS (2000) 97:10526; Erbayraktar et al., PNAS (2003) 100:6741; Genc et al., Neurosci. Lett. (2001) 298:139; Konishi et al., Brain Res. (1993) 609:29; Puskovic et al., Mol. Ther. (2006) 14:710; Sakanaka et al., PNAS (1998) 95:4635; Bartels et al., Ther Adv Neurol Disord. (2008) 1:193; Sargin et al, Best Pract Res Clin Anaesthesiol. (2010) 24:573; Juul, J Matern Fetal Neonatal Med. (2012) Suppl 4:97). Because of their broad protective activities, EPO and EPO mimetics are promising agents also for the treatment of diabetes mellitus (Choi et al., Curr Diabetes Rev. (2011) 7:284).
EPOR is abundantly expressed in the CNS, including neurons, astrocytes and choroid plexus epithelial cells (Bernaudin et al., J. Cereb. Blood Flow Metab. (1999) 19:643; Digicaylioglu et al., PNAS (1995) 92:3717; Sirén et al., Acta Neuropathol. (2001) 101:271). The presence of EPOR is critical for early embryonic neural development as well as for adult neurogenesis and migration of regenerating neurons during post-stroke recovery (Tsai et al., J. Neurosci. (2006) 26:1269). While the expression of EPOR decreases in normal adult neural tissues (Liu et al., J. Biol. Chem. (1997) 272:32395), the expression of both EPO and its receptor is upregulated following ischemia, supporting a critical role for the EPO/EPOR system in protection against ischemic brain injury (Bernaudin et al., J. Cereb. Blood Flow Metab. (1999) 19:643). EPO and EPO mimetics are also implicated to have beneficial protective effects in glaucoma, optic nerve injury, age-related macular degeneration, chronic diabetic macular edema, retinopathy, peripheral nerve injury and in peripheral neuropathy associated with for example diabetes or chemotherapy (Bianchi, Eur J Cancer. (2007) 43:710; Tsai, J Glaucoma. (2007) 16:567; Takagi, Diabetes Res Clin Pract. (2007) 77 Suppl 1:S62; Schmidt, Exp Neurol. (2008) 209:161; Wang et al., Chin Med J (Engl). (2009) 122:2008; Wang et al., Med Hypotheses. (2009) 72:448; Li et al., (2010) Ophthalmic Surg Lasers Imaging. 41:18; Yin, et al., AJNR Am J Neuroradiol (2010) 31:509). However, pharmacokinetic properties of rhEPO are not well suited for CNS diseases. The concentrations of rhEPO in human cerebrospinal fluid were generally more than 1000-fold lower than in circulation following intravenous administration (Xenocostas et al., Eur. J. Clin. Pharmacol. (2005) 61:189).
EPO and EPO Mimetics in Ischemic Stroke and Brain Injury
Initial indications of EPO's cytoprotective effects in vivo were derived from studies in adult rats that had undergone fimbria-fornix transections that indicated enhanced survival of septal cholinergic neurons following rhEPO treatment (Konishi et al., Brain Res. (1993) 609:29). In addition, infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from ischemia-induced cell death (Sakanaka et al., PNAS (1998) 95:4635). These results were corroborated in hippocampally injured rats, in which rhEPO administration associated with a virtually complete elimination of the otherwise severe behavioral impairment caused by fimbria-fornix transection (Malá et al., Neural Plast. (2005) 12:329).
Systemically administered rhEPO induces significant protective effects in preclinical models of focal brain ischemia and brain injury, albeit upon frequent administration of large doses of rhEPO. RhEPO reduced infarct volume by approximately 50-75% in a rat model of MCA occlusion (Brines et al., PNAS (2000) 97:10526). Dose-response studies determined that three doses of 5,000 and one dose of 30,000 U/kg rhEPO are protective in seven-day-old rats undergoing unilateral carotid ligation plus 90 minutes of 8% hypoxia (Kellert et al., Pediatr Res. (2007) 61:451). RhEPO treatment also promotes angiogenesis, improves the growth of microvessels, and restores local cerebral blood flow after embolic stroke or permanent focal cerebral ischemia in rats and mice (Li et al., J. Cereb. Blood Flow Metab. (2007) 27:1043). RhEPO treatment significantly reduced endothelial cell death and increased neuronal cell proliferation following focal ischemic stroke in neonatal rats (Keogh et al., J. Pharmacol. Exp. Ther. (2007) 322:521). Both rhEPO and carbamylated EPO had anti-inflammatory and anti-apoptotic effects and resulted in neurologic improvement when administered six hours following embolic MCA occlusion in rats (Wang et al., Br. J. Pharmacol. (2007) 151:1377. Mice treated with daily intraperitoneal injections of a 13-aa EPO peptide exhibited an improvement in neurologic score on day three following MCA occlusion when compared to mice treated with saline (Yuan et al., WO 2007/052154). The same peptide also appeared protective in a mouse model of contusive spinal cord injury.
The beneficial role of rhEPO and other EPOR Agonists has also been demonstrated in several models of traumatic brain injury. For example, rhEPO protected rats from closed head injury (Yatsiv, Faseb J (2005) 19, 1701), blunt trauma caused by a pneumatic piston (Brines, PNAS (2000) 97:10526, radiosurgery (Erbayraktar, Mol Med (2006) 12:74), controlled cortical impact injury induced by gas pressure (Cherian, J Pharmacol Exp Ther (2007) 322:789), and fluid percussion (FP) injury (Hartley, J Neurosurg (2008) 109:708). RhEPO was also shown to induce neuroregeneration following traumatic brain injury, which was associated with enhanced expression of brain-derived neurotrophic factor (BDNF) in vivo (Mahmood et al., J. Neurosurg. (2007) 107:392).
Systemic rhEPO was shown to reduce delayed cerebral ischemia and improve outcome scores following aneurysmal subarachnoid hemorrhage in an 80-patient Phase II randomized, doubled-blinded placebo-controlled trial (Tseng et al., J Neurosurg (2009) 111:171). Moreover, repeated low-dose rhEPO improved neurological outcome and reduced the risk of disability in a study of 153 infants with hypoxic-isehemic encephalopathy when compared to placebo (Zhu, Pediatrics (2009) 124:e218).
EPO and EPO Mimetics in Parkinson's Disease (PD)
Several studies have shown a direct benefit of neuroprotection from exogenous EPO receptor agonists on the motor behavior in rodent models of PD (Gene et al., Neurosci. Lett. (2001) 298:139; Kanaan et al., Brain Res. (2006) 1068:221). Direct neuroprotective effects of EPO have been shown using both primary dopaminergic neurons and dopaminergic cell lines. EPO induces neuroprotective effects through inhibition of apoptosis, which involves activation of the Jak2/STAT5 signaling pathway, upregulation of Bcl-2 and Bcl-xL, activation of Akt/GSK signaling, and downstream reduction of caspase-3 activity (Wu et al., Apoptosis. (2007) 12:1365; Silva et al., Blood (1996) 88:1576). Small molecule EPO mimetics are expected to provide significant benefits over protein- and peptide-based EPO receptor agonists through improved crossing of the BBB, oral dosing and lack of immunogenicity, and such compounds will have significant potential as disease modifying agents in PD.
EPO and EPO Mimetics in Cognitive Function and Alzheimer's Disease (AD)
EPO Receptor Agonists are promising in the treatment of AD. In 4-week old mice with unilateral parietal cortex injury, a model for early aging and neurodegeneration, EPO treatment given for 2 weeks immediately following injury prevented behavioral abnormality, cognitive dysfunction and brain atrophy seen in control mice. The results were independent of hematopoietic effects (Siren et al., Brain (2006) 129: 480). A related study in young mice without parietal injury showed that EPO treatment every other day for three weeks improved hippocampal dependent memory compared to untreated controls (Adamcio et al., BMC Biol. (2008) 6:37).
EPO's indirect neuroprotective effects have also been studied in animal models in vivo in the context of other neurotrophic factors. EPO and CEPO were shown to increase BDNF in rats in the dentate gyrus, part of the hippocampus related to spatial learning in traumatic brain injury (Mahmood et al., J. Neurosurg. (2007) 107:392). In addition, EPO increased BDNF mRNA expression in whole brain 1 hour following intracerebroventricular injection in mice (Viviani et al., J Neurochem. (2005) 93:412). BDNF and its receptor TrkB are expressed in the entorhinal cortex, which is involved in neurodegeneration and short-term memory loss early in AD. BDNF also prevented lesion-induced death of cortical neurons and reduced cognitive impairment in primates (Nagahara et al., Nat Med. (2009) 15:331), further suggesting that the effects of EPO on BDNF expression may have important beneficial effects in AD and other neurodegenerative diseases.
EPO and EPO Mimetics in Friedreich's Ataxia (FRDA)
Friedreich's ataxia (FRDA) is an inherited neurodegenerative disease primarily caused by a GAA-trinucleotide repeat expansion in the first intron of the frataxin gene (FXN), resulting in reduced frataxin protein expression (Campuzano et al., Science (1996) 271:1423). Frataxin protein levels in FRDA patients vary between 4% and 29% of healthy individuals, while carriers have a 50% reduction of frataxin expression and are asymptomatic. EPO and EPO mimetics are promising agents for the treatment of FRDA because of their neuro- and tissue-protective effects and because they enhance the levels of frataxin protein. In preclinical and clinical studies, rhEPO and other EPOR Agonists, such as carbamylated EPO, increased frataxin expression in both normal and patient cells in vitro and in vivo. RhEPO was shown to increase frataxin levels in vitro in several cells including P19 neurons, FRDA lymphocytes, primary human cardiac myocytes and fibroblasts (Sturm et al., Eur J Clin Invest. (2005) 35:711). In an open label pilot trial with 8 FRDA patients, rhEPO demonstrated an overall 24% increase in frataxin in lymphocytes with a significant improvement in ataxia rating scores (FARS and SARA) and indicators of oxidative stress that persisted during 6 months of treatment (Boesch et al., Mov Disord. (2008) 23:1940). In addition, in patients with chronic kidney disease, rhEPO infusion for anemia caused an increase in frataxin levels by 2-3-fold in peripheral blood mononuclear cells (Sturm et al., Eur J Clin Invest. (2005) 35:711; U.S. Pat. No. 7,790,675). Erythropoietic activity of rhEPO is not required for neuroprotection or frataxin increase, because EPO derivatives without hematopoietic activity enhance neuroprotection and frataxin levels both in vitro and in vivo (Brines et al., PNAS (2008) 105:10925; Sturm et al., Eur J Clin Invest. (2010) 40:561).
EPO and EPO Mimetics in Depression, Schizophrenia, Drug Abuse and Addiction
EPO mimetics have the potential for improvement of cognitive function, mood and overall quality of life. EPO mimetics also have potential in the treatment of addiction and substance abuse, in part due to EPO's ability to upregulate BDNF and GDNF levels. In addition, EPO has been implicated to have potential in the treatment of attention deficit hyperactivity disorder (McPherson, Int J Dev Neurosci. (2008) 26:103).
Low BDNF levels have been associated with depression, bipolar disorder, chronic heroin use and alcohol dependence (Joe, Alcohol Clin Exp Res. (2007) 31:1833; Angelucci, et al., J Psychopharmacol. (2007) 21:820; Tseng et al., J Psychiatry Neurosci. (2008) 33:449; Umene-Nakano et al., Hum Psychopharmacol. (2009) 24:409). In addition, polymorphisms in the BDNF gene associates with bipolar disorder, schizophrenia, alcohol dependence and tobacco smoking (Neves-Pereira et al., Am J Hum Genet. (2002) 71:651; Neves-Pereira et al., Mol Psychiatry (2005) 10:208; Matsushita et al., Alcohol Clin Exp Res. (2004) 28:1609; Novak et al., Ann Hum Genet. (2010) 74:291). Given that EPO and EPO-R agonists have been shown to enhance BDNF expression in vitro and in vivo (Mahmood et al. J. Neurosurg. (2007) 107:392; Viviani et al., J Neurochem. (2005) 93:412), EPO mimetic compounds have potential in the treatment of these disorders. Similarly, GDNF levels were elevated in the brain following EPO-treatment (Dzietko, Neurobiol Dis. (2004) 15:177), and GDNF signaling has been implicated in the treatment of drug and alcohol addiction (Ron, Rev Neurosci. (2005) 16:277). Transplantation of GDNF-expressing cells into the striatum and nucleus accumbens was shown to attenuate acquisition of cocaine self-administration in rats (Green-Sadan et al., Eur J Neurosci. (2003) 18:2093).
The beneficial effects of EPO on cognitive function and mood, combined with enhanced expression of both BDNF and GDNF support the notion that EPO mimetic compounds have potential in the treatment of several psychological disorders, including conditions with reduced expression of BDNF and GDNF. Three-day treatment with rhEPO improved cognitive and neural processing of happy and fearful faces and improved self-reported mood for all three days following administration in healthy volunteers (Miskowiak, J. Neurosci. (2007) 27:2788; Miskowiak, Biol. Psychiatry (2007) 62:1244). In addition, rhEPO increased hippocampus response during picture retrieval, further suggesting potential in the treatment of psychiatric disorders (Ehrenreich, Mol. Psychiatry. (2004) 9:42; Miskowiak, Biol. Psychiatry (2007) 62:1244). Furthermore, schizophrenic patients treated with rhEPO demonstrated a significant improvement over placebo in schizophrenia-related cognitive performance (RBANS subtests, WCST-64) (Ehrenreich, Mol Psychiatry (2007) 12:206). RhEPO and other EPOR Agonists are also promising in the treatment of depression, which is frequently comorbid with for example alcohol dependence and pathological gambling (Umene-Nakano et al., Hum Psychopharmacol. (2009) 24:409; Romer Thomsen et al., Behav Pharmacol. (2009) 20:527).
EPO and EPO Mimetics in Spinal Muscular Atrophy and Amyotrophic Lateral Sclerosis
Spinal muscular atrophy is a recessively inherited neurodegenerative disease that is characterized by mutations in the survival motor neuron (SMN) gene, reduced levels of functional SMN protein, and resulting motor neuron death (Schrank et al., PNAS (1997) 94:9920). Clinical symptoms include progressive muscular atrophy and premature death. EPO mimetics have demonstrated significant neuroprotection, and therefore, would be expected to reduce neurodegeneration or increase neuroregeneration leading to an effective disease modifying treatment.
Amyotrophic lateral sclerosis (ALS) is an inherited neurodegenerative disease that is characterized by a progressive loss of motor neurons that control voluntary muscle movement. Recombinant EPO reduced symptoms and improved secondary survival signals in a mouse model of the disease and is being tested in patients (Koh et al., Eur J Neurosci. (2007) 25:1923; Lauria et al., Amyotroph. Lateral Scler. (2009) 10:410). Therefore, EPO mimetics are also expected to reduce neurodegeneration or increase neuroregeneration leading to an effective treatment for ALS.
EPO and EPO Mimetics in Transplantation
The tissue- and neuroprotective effects of rhEPO and other EPOR Agonists are beneficial also when developing means to enhance engraftment and survival in tissue and organ transplants. EPO was shown to improve liver regeneration and survival in rat models of extended liver resection and living donor liver transplantation (Bockhorn et al., Transplantation. (2008) 86:1578). High dose recombinant EPO, nonerythropoietic EPO derivatives in particular, are promising in prevention of kidney damage and loss of renal function after successful kidney transplantation (van Rijt et al., Transpl Int. (2013) Aug. 5 [Epub ahead of print]). The neuroprotective and neuroregenerative effects of EPO and EPO mimetics have the potential to further enhance the functionality of the transplanted tissue, for example in the case of skin transplantation to enhance the recovery of sensation in the transplanted tissue.
EPO and EPO Mimetics in Organ Protection and Tissue Repair
A significant role of rhEPO and other EPOR Agonists in tissue repair has emerged, suggesting therapeutic applications for indications such as wound healing (Galeano et al., Diabetes. (2004) 53:2509, Galeano et al., Crit. Care Med. (2006) 34:1139), cardioprotection following ischemia (Fiordaliso et al., PNAS (2005) 102:2046), kidney protection (Bahlmann et al., Circulation (2004) 110:1006; Tillmann et al., Kidney Int. (2006) 69:60) and liver protection and repair (Shawky et al 2012). In mice, EPO treatment enhances early endochondral ossification and transition from soft callus to hard callus, suggesting a role for EPO in bone repair and fracture healing (Holstein et al., Life Sci (2007) 80:893).
EPO and EPO Mimetics in Stem Cell Therapies
An EPO mimetic can also improve stem cell therapies due to its multiple activities on cellular functions in different tissues. The ability to generate pluripotent human stem cells from adult fibroblasts has significantly enhanced the potential of stem cell-based therapies as a practical approach for clinical indications (Takahashi et al., Cell (2007) 131:861; Yu et al., Science (2007) 318:1917). EPO receptor agonist compounds enhance oxygenation and expansion of stem cells, and, thereby, improve development of therapies for indications where stem cell-based approaches have demonstrated promising results, such as myocardial infarction, soft-tissue injury, heart failure, repair of atherosclerotic vessels and diseases of the central nervous system (Sylvester and Longaker, Arch. Surg. (2004) 139: 93; Urbich and Dimmeler, Circ. Res. (2004) 95: 343; Yoon et al. Biol Cell. (2005) 97: 253; Martino and Pluchino, Nat. Rev. Neurosci. (2006) 7: 395). EPO mimetic compounds with improved solubility and permeability would offer advantages in cell-based therapies, such as the improved distribution in wounded tissue and enhanced engraftment of transplanted cells.
EPO and EPO Mimetics in Production of Red Blood Cells (RBC) Ex Vivo
EPO and EPO mimetics play a significant role in expanding stem cells and RBC ex vivo for the benefit of cell transplantation and transfusion medicine. Neildez-Nguyen et al. described a protocol for the significant expansion of CB-derived CD34+ hematopoietic progenitor cells in a well-defined serum-free medium in the absence of feeder cells and stroma (Neildez-Nguyen et al., Nat. Biotechnol. (2002) 20: 467). Based on the sequential addition of growth factors, including TPO, FL, KL, EPO and IGF-1, this protocol allowed for a 200,000-fold amplification of pure erythroid progenitors. When administered to immunodeficient (NOD-SCID) mice, these progenitors continued to proliferate in vivo and differentiated within four days to the terminal stage of enucleated cells producing adult Hb (Neildez-Nguyen et al., Nat. Biotechnol. (2002) 20: 467). When stromal cell were included in the culture protocols, 100% terminal differentiation into mature RBC was observed and associated with >200,000-fold amplification of CD34+ progenitors (Giarratana et al., Nat. Biotechnol. (2005) 23: 69). Cultured RBC generated using this method demonstrated almost identical maturity, functionality, and half-life when compared to natural RBCs in vitro and in vivo, including in a clinical trial (Giarratana et al., Blood (2011) 118:5071).
One approach to improve the efficiency and reduce the cost of cultured RBC is to utilize small molecule cytokine mimetics. Small molecules have relatively longer shelf-life and do not require cold-chain infrastructure for storage, in contrast to most recombinant proteins.